Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/82—Proteins from microorganisms
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/82—Proteins from microorganisms
  • Y10S530/823—Lower fungi, e.g. mold

Definitions

• the present invention relates to microorganisms belonging to the genus Humicola, in particular, Humicola 'insolens
• Filamentous fungi are known to secrete proteins extracellularly. Therefore, with regard to filamentous fungi, high-producing cells and culture methods for efficiently producing proteins have been studied and developed.
• the main method is to create artificial mutations by UV irradiation, mutagenic agents, etc., and select a strain that produces a large amount of the target protein from them.
• thermophilic filamentous fungus Humicola insolens also has excellent protein secretion ability. It is also known that Humicola insolens produces a variety of industrially useful cellulases (W091 / 17,243, JP-T-5-509,223). ).
• the useful component in the total secreted protein of Shimadori, Shinaikikura and Humicola insolens is only a few percent. Establishing a method to express and secrete large amounts of these trace useful components will dramatically improve the performance of the final product. In addition, if a method for expressing large amounts of heterologous genes in Humicola insolens is established, various enzymes and useful proteins can be produced by a single method, which will result in a cheaper supply capacity.
• Humicola insolens as a useful protein production host is a thermophilic filamentous fungus, so the optimal culture temperature is around 37 ° C, and it is less susceptible to contamination by other bacteria during culture. There are advantages.
• the present inventors have now established a method for mass-producing a substance belonging to the genus Humicola, particularly a target protein in Humicola insolens.
• an object of the present invention is to provide a substance belonging to the genus Humicola, particularly an expression system which enables large-scale production of proteins in Humicola insolens, particularly a host-to-host system, and a method for producing a protein using the same.
• an extremely efficient protein production system in which the productivity of the target protein is 10 to 16 times that of the parent strain and the production amount is about 4.5 g per liter of the culture solution. Is done.
• FIG. 1 is a restriction enzyme diagram of plasmid pM3_1.
• Figure 2 shows the restriction map of plasmid pMl4-1.
• FIG. 3 is a restriction map of the plasmid vector pMKDO1.
• FIG. 4 is a restriction map of the plasmid vector pEGDO1.
• FIG. 5 is a restriction map of the plasmid vector pIED02.
• the Escherichia coli J M109 strain transformed with the plasmid pM3-1 represented by the map shown in Fig. 1 was FERM BP-5971 (Original deposit: FE RM P-14459, Original deposit date: August 1994) It has been deposited with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Life Science and Industrial Technology Research Institute (1-1-3, Higashi, Tsukuba, Ibaraki, Japan) under the accession number 3).
• the strain JM109 transformed with the plasmid pMl4-1 represented by the map shown in FIG. 2 was FERM BP-5972 (original deposit: FE RM P-1458). 5, Original deposit date: October 18, 1994), deposited under the Ministry of International Trade and Industry's Industrial Technology ⁇ Life Engineering Industrial Technology Research Institute under the accession number.
• the E. coli JMl09 strain transformed with the expression vector pMKDO1 according to the present invention was traded under the accession number of FERM BP-5974 (original deposit: FERM P-15730, current deposit date: July 12, 1996). It has been deposited at the Institute of Biotechnology and Industrial Technology, the Ministry of Industry.
• the Escherichia coli J Ml09 strain transformed with the expression vector pEGDO1 according to the present invention was traded under the accession number of FERM BP-5973 (Original deposit: FERM P-15729, Original deposit date: July 12, 1996). It has been deposited at the Institute of Biotechnology and Industrial Technology, the Ministry of Industry.
• the E. coli JMl09 strain transformed with the expression vector p I EDO2 according to the present invention has the accession number of FERM BP-5975 (original deposit: FERM P-15731, original deposit: July 12, 1996). And deposited at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.
• the E. coli JM109 strain transformed with the expression vector pNCE4Sa1 according to the present invention was deposited under FERM BP-5976 (original deposit: F ERM P-15732; Hara deposit: July 12, 1996). It is deposited at the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry under the number.
• Humicola insolens MN 200-1 which can be a host of the expression vector according to the present invention, was traded under the accession number of FERM BP-5977 (Original deposit: F ERM P-15736, Original deposit date: July 15, 1996). Ministry of Industry, Technology and Technology Deposited with the Institute of Technology.
• proteins and peptides are used synonymously unless otherwise specified.
• the modified sequence refers to a base sequence or an amino acid sequence.
• amino acid sequence it means that one or several bases or amino acids have been inserted, substituted or deleted, or 3 ⁇ 4 has been added to one or both ends thereof.
• control sequence derived from Humicola insolens is used.
• the control sequence means at least one selected from the group consisting of a promoter, a signal sequence, and a terminator.
• the control sequence according to the present invention is preferably a control sequence of the cellulase NCE1 gene derived from Humicola insolens described in JP-A-8-56663, and furthermore, a Humicola 'insolenz described in JP-A-8-126492.
• This is a regulatory sequence of a cellulase NCE2 gene derived therefrom. More specifically, NCE 1 and 2 within plasmids pM3-1 and pMl4-1 in the strains deposited under FERM BP-5971 and FERM BP-5972. This is the control array.
• Examples of preferred promoter sequences in the present invention include a sequence present in a region from the N-terminus of the NCE1 gene up to about 1500 bP in the plasmid pM3-1 shown in the map of FIG. The sequence from the N-terminus of NCE 1 site to the BgIII site upstream is shown in the figure.
• a preferred promoter sequence in the present invention is a plasmid pMl4-1 shown in the map of FIG. 2 in the region from the N-terminus of the NCE2 gene up to about 1500 bp upstream.
• the sequence from the N-terminus of the NCE2 gene to the upstream EcoRI site in the figure is a plasmid pMl4-1 shown in the map of FIG. 2 in the region from the N-terminus of the NCE2 gene up to about 1500 bp upstream.
• the promoter sequence according to the present invention includes not only the entire sequence of these regions but also its modified sequence that retains high promoter activity.
• high motor activity means high expression in NCE4 gene expression described below. More specifically, a promoter activity that expresses 2.Og, preferably 4.Og, more preferably 4.5g of NCE4 per liter of medium.
• examples of a preferable signal sequence in the present invention include a signal sequence of cellulase NCE 1 or 2. More specifically, a sequence encoding the sequence of amino acids 22 to 11 of the amino acid sequence of SEQ ID NO: 1; and the sequence of amino acids 23 to 1 of the amino acid sequence of SEQ ID NO: 2 Code the array ⁇ Arrays are listed.
• the invention further includes modified sequences thereof that encode an amino acid sequence that still retains signal sequence activity. Such modified sequences are also described in the Examples described below, the strains deposited under FERM BP-5971 and FER BP-5972, and those skilled in the art given the mapping ability of FIG. 1 or 2. If so, it is clear that the presence of such a modified sequence can be easily predicted, and that it can be easily produced.
• the target protein is obtained as a fusion protein with a peptide consisting of several amino acids at the N-terminal side of NCE1 or NCE2, and further as a fusion protein with NCE1 or NCE2. You may be.
• a preferred terminator sequence in the present invention is the plasmid pM3-1 shown in the map of FIG. Sequences existing in a region of up to 00 bp, for example, a sequence from the C-terminus of NCE ⁇ to a downstream Bgill site. Another example is a sequence present in a region from the C-terminus of NCE 2 ⁇ to about 500 bp downstream of NCE 2 ⁇ in the plasmid pM14-1 shown in the map of FIG. 2, for example, the NCE 1 gene. From the C-terminus to the downstream Bg1II site.
• the terminator sequence according to the present invention includes not only the entire sequence of these regions but also a modified sequence that retains the terminator activity.
• control sequence preferably used for expression of the NCE4 gene is 10 to 16 times that of the parent strain from which the enzyme is derived, and the production amount is 1 liter of culture solution. Each reaches 2.0 g, preferably 4.0 g, more preferably about 4.5 g.
• Expression vector and host is 10 to 16 times that of the parent strain from which the enzyme is derived, and the production amount is 1 liter of culture solution. Each reaches 2.0 g, preferably 4.0 g, more preferably about 4.5 g.
• an expression vector for expressing a target protein using the control sequence is provided.
• the expression vector according to the present invention comprises the above-mentioned control sequence and, in some cases, a gene marker. Furthermore, an expression vector according to the present invention comprises the expression vector of the first aspect, and further comprises a base sequence encoding a target protein operably linked to its control sequence. Therefore, an expression vector comprising at least one selected from the group consisting of the above-mentioned promoter, signal sequence, and terminator according to the present invention is included in the scope of the present invention.
• the promoter sequence according to the present invention has extremely high utility.
• an expression vector comprising at least the promoter sequence of the present invention.
• the signal sequence and the terminator sequence may be other than the signal sequence and the terminator sequence according to the present invention. Use is preferred.
• Specific examples of these vectors include the expression vectors pMKD01, pEGD01, and p1ED02 constructed in Examples described later, in which the NEC3 and NCE4 genes are removed. Is mentioned.
• the expression vector according to the present invention is preferably constructed on the basis of a vector capable of replicating in a host cell, for example, a plasmid.
• a vector capable of replicating in a host cell for example, a plasmid.
• examples of such vectors include pUC Vector, pTV Vector. PBluescript, and pBR322, which are vectors that can be replicated in E. coli.
• a technique required for constructing the vector according to the present invention a method commonly used in the field of gene recombination can be used.
• the gene may be appropriately selected depending on the method of selecting a transformant.
• a gene encoding drug resistance and a gene complementing auxotrophy can be used.
• the drug resistance gene used in the present invention is not limited as long as it relates to a drug to which a host cell is sensitive.
• a destomycin-resistant gene derived from Streptomyces rimofaciens, Escherichia E. coli-derived digromycin B resistance gene, Streptococcus hi ndustanus-derived bleomycin resistance gene, and Streptomyces hygroscopicus-derived bialaphos resistance gene can be preferably used.
• the promoter and terminator of the trp C gene derived from Aspergillus niduians are obtained by a known method.
• the ability to use a cassette which is capable of expressing a destomycin resistance gene Japanese Unexamined Patent Publication (Kokai) No. 59-175889) is preferably used.
• the expression vector according to the present invention can be used for expression production of various target proteins or peptides.
• the target protein or peptide means not only a so-called foreign protein that is not present in Humicola insolens, but also a protein that is expressed in Humicola insolens but is a quantity thereof.
• the residue encoding the target protein in the expression vector according to the present invention includes a residue encoding an industrially useful protein such as cellulase, amylase, lipase, lipase, phytase and the like. . Genes obtained by artificially improving these can also be used as target proteins.
• the expression vector according to the present invention is used as an expression system in combination with a host belonging to the genus Humicola as a host.
• a host belonging to the genus Humicola as a host.
• Humicola insolens is used as a substance belonging to the genus Humicola.
• the expression system according to the present invention can be preferably used for producing cellulase NCE4 derived from Humicola insolens or a modified protein thereof as a target protein.
• the cellulase NCE4 derived from Humicola insolens means the protein described in SEQ ID NO: 3. This is a cellulase enzyme isolated by the present inventors, as described in Examples below.
• the modified protein is a protein in which amino acid addition, insertion, deletion of IJ, deletion, or substitution has occurred in the amino acid sequence of the above protein, and is still the same as cellulase NCE4. That retain cellulase activity, especially endoglucanase activity.
• an expression vector constructed according to an example described later is used as a specific example of a vector preferable as an expression system for these cellulase NCE4.
• an expression vector constructed according to an example described later is used as a specific example of a vector preferable as an expression system for these cellulase NCE4.
• Transformation of host cells with the expression vector according to the present invention can be carried out by a method commonly used in the field of genetic recombination, for example, according to the method described in JP-A-8-56663. Can be implemented. It may also be implemented by electroporation.
• the production of the target protein according to the present invention is carried out by culturing a host cell transformed with the above-described expression vector according to the present invention in a suitable medium, and collecting the target protein or the peptide from the culture. .
• the productivity of the target protein is 10 to 16 times that of the parent strain, and the production amount is 2.0 g, preferably 4.0 g, per liter of culture.
• a very efficient protein production system more preferably up to about 4.5 g, is provided.
• the host cell is Humicola 'insolens, it can produce 2.O g, preferably 4.O g, more preferably 4.5 g or more of the target protein per liter of the culture.
• This amount is extremely large as compared with conventionally known protein expression systems. This indicates that the expression system of the target protein according to the present invention has extremely high utility.
• the protein of interest is cellulase NCE3 or NCE4, these enzymes are inherently highly active, but can produce them in larger quantities. As a result, there is obtained an advantage that it is possible to efficiently produce a cellulase preparation useful for removing fluff of cellulose-containing fibers, reducing the weight thereof, and decolorizing denim-dyed cellulose-containing fibers.
• the transformant is cultured in a liquid medium containing conventional components such as a carbon source, a nitrogen source, an inorganic salt, and a growth factor component under aerobic conditions, Perform by shaking culture, electric stirring culture or submerged culture be able to.
• the pH of the medium is, for example, about 7 to 8.
• Culturing is carried out under the usual conditions customary for culturing Humicola insolens, for example when the host cell is Humicola insolens, for example 15 ° C to 45 ° C, preferably 35 to 40.
• the cultivation time can be about 24 to 240 hours.
• ordinary separation means utilizing the properties thereof, such as a solvent extraction method, an ion exchange resin method, an adsorption or distribution column chromatography method, and a gel filtration method , Dialysis, precipitation, etc. can be used alone or in appropriate combination.
• Example A 1 Isolation and Purification of a Component Active in Removing Tencel Fuzz from Humicola Insolens
• Humicola Insolens MN 200-1 in (N) medium (5.0% Avicel, 2.0% yeast extract, 0.1% polypeptone, 0.03% calcium chloride, 0.03% magnesium sulfate, pH 6.8 ), And cultured at 37 ° C. After culturing for 7 days, the resulting culture was centrifuged at 7000 rpm for 20 minutes to remove the cells, and the culture supernatant was used as a crude cellulase preparation.
• This crude cellulase preparation was subjected to hydrophobic mouth chromatography (Pheny Sepharose High Performance 6/100, manufactured by Pharmacia Biotech) and subjected to a concentration gradient of 110 M in 50 mM phosphate buffer (pH 7.0). Eluted with the ammonium sulfate solution and fractionated. Among them, the fraction obtained at a concentration gradient of 0.1 to 0 M showed strong activity to remove Tencel fuzz, and the fraction was again subjected to hydrophobic chromatography (Phenyl-Sepharose High Performancel 6/100). Ammonium sulfate in a concentration gradient of 0.4-0 M in 5 OmM phosphate buffer (pH 7.0) The active fraction was eluted with the solution and collected.
• the fraction was subjected to reverse phase chromatography (Sourcel5 IS0, manufactured by Falman Biotech), and ammonium sulfate subjected to a concentration gradient of 110 M in 5 OmM phosphate buffer (pH 7.0). Eluted with the solution and fractionated. Among them, the fraction obtained at 0M portability showed strong tencel fuzz removal activity, and the fraction was subjected to reverse phase chromatography (Source 15 PHE, manufactured by Pharmacia Biotech). Elution with ammonium sulfate solution with a concentration gradient of 1 to 0 M in 5 OmM phosphate buffer (pH 7.0) was performed, and the fraction with high tencel fuzz removal activity was isolated as a purified enzyme NCE 4. did. This NCE 4 showed a single band with a molecular weight of 43 kDa in SDS-PAGE.
• N-terminal amino acid sequence Ala-Asp-Gly-Lys-Ser-Thr-Arg-Tyr-Trp-Asp- (Cys)-(Cys) -Lys-Pro-Ser (15 residues)
• the protein purified by the FPLC of the above (1) was lyophilized and then dissolved in 100 mM ammonium bicarbonate buffer (pH 8.0). About 1 to 20 moles of trypsin (Promega) was added to the protein and reacted at 37 ° C for 48 hours. This degradation product was subjected to column chromatography using a Model 172 preparative HPLC system (manufactured by PerkinElmer) (column: C8220 2.1 mm, 0.1% TFA, 0% acetonitrile to 0.085% TFA, 35% acetonitrile gradient) and three kinds of peptides were collected. The amino acid sequence of the obtained peptide fragment was determined by the aforementioned protein sequencer. The results were as follows.
• the N-terminal amino acid sequence and the amino acid sequence obtained by peptide mating were obtained from the 43KDa endoglucanase obtained from Humicola insolens DSM1800 described in W091Z17243 (Tokuhyo Hei 5-509223).
• the protein showed homology to the amino acid sequence of the above, strongly suggesting that the protein was a kind of cellulase.
• Genomic DNA was isolated according to the method of Horiuchi et al. (Hiroyuki Horiuchi et al., J. Bacteriol., 170: 272-278, 1988).
• Humicola insolens MN200-1 was cultured at 37 ° C. in the aforementioned (N) medium. After culturing for 2 days, cells were collected by centrifugation (3500 rpm, 10 minutes). The obtained cells were treated with phenol, treated with proteinase and ribonuclease A, and further subjected to polyethylene glycol (PEG) precipitation to obtain genomic DNA.
• N aforementioned (N) medium.
• PEG polyethylene glycol
• Humicola insolens genomic DNA was digested with Sau3A I, and after agarose gel electrophoresis was confirmed to be partially degraded in the range of 9 to 23 kbp, this was recovered by ethanol precipitation.
• This DNA fragment was ligated to a BamHI arm of a phage vector, EMBL3 cloning kit (Stratagene) using T4 ligase (Toyobo Co., Ltd.). After precipitation with ethanol, this was dissolved in a TE (1 OmM Tris-HCl (pH 8.0), lmM EDTA) buffer solution.
• the entire ligation mixture was frozen and packaged as described in the method of Hohn. B. (Hohn, B. Methods Enzymol., 68: 299-309, 1979) and the Gigapack II Packaging Kit (Stratagene). Phage), and the resulting phage was used to infect E. coli strain LE392. Using the 5 ⁇ 1 CT phage library obtained by this method, the target gene was cloned.
• a long probe was prepared by PCR using the total DNA of Humicola 'insolens type II and used. Each primer synthesized DNA corresponding to the amino acid indicated by * at the N-terminus and the peptide TP-3.
• the sequence of the prepared synthetic oligonucleotide was as shown below.
• NCE4N1 5'-GCXGA (CT) GGXAA (AG) TC (AGCT) AC-3 '(17mer)
• NCE4N2 5 'GCXGA (CT) GGXAA (AG) AG (CT) AC-3' (17mer)
• NCE4C 5- 'CXGC (AG) TT (CT) TT (AG) AACCA (AG) TC-3' (19mer)
• the PCR reaction was performed under the following conditions. First, two sets of tubes were added to the Humicola insolens genome D NAl zg, with 1 M each of NCE4N1 and NCE4C added as primers, and 1 M each of NCE4 N2 and NCE4C added as primers. Heat denaturation was performed at 5 ° C for 5 minutes. Thereafter, Taq polymerase (Recombinant Taq, manufactured by Takara Shuzo) was added, and amplification was performed by repeating the reaction conditions of 94 ° C for 1 minute, 45 ° C for 2 minutes, and 72 ° C for 3 minutes 25 times. As a result, about 750 bp of DNA was amplified only when NCE4NK NCE4C was used as a primer. This was used as a probe for subsequent screening.
• Taq polymerase Recombinant Taq, manufactured by Takara Shuzo
• the phage plaques prepared according to the method described in Example 2 were transferred to Hybond Nylon Trans-Farm Membrane (manufactured by Amersham), denatured with 0.4N sodium hydroxide, and then denatured with 5-fold concentration SSC (15 mM The mixture was washed with trisodium acid (15 OmM sodium chloride), dried, and the DNA was fixed. Following the kit procedure, pre-hybridization (42 ° C) for 1 hour, followed by labeling The probe thus obtained was added, and hybridization was performed for 4 hours (42.C). Label washing was performed according to the kit method described above. First, wash twice for 20 minutes at 42 ° C with 0.5% SSC containing 0.4% SDS and 6 M urea, and then wash twice for 5 minutes at room temperature with 2% concentration SSC. Was.
• the membrane was immersed in the attached detection solution for 1 minute and exposed to Hyperfilm-ECL (Amersham) to obtain 4 positive clones.
• E. coli LE 392 was infected with phage, and after 8 hours, phage particles were collected and treated with proteinase K and phenol by Grossberger's method (Grossberger, D., Nucleic Acids. Res. 156737.1987), followed by ethanol precipitation. Phage DNA was isolated.
• the four phage DNAs were digested with SalI and subjected to agarose electrophoresis.
• the DNA was transferred to a nylon membrane by the Southern method (Southern, EM, J. Mol. Biol. 98: 503-517, 1975), under the same conditions as the plaque hybridization of the previous method (1). Then, hybridization was performed using a probe of about 750 bp, and a DNA fragment containing the 5.2 kbp target gene was detected. As a result, four kinds of phage DNAs had Sal I fragments of the same size.
• the nucleotide sequence was determined as follows.
• A.L.F. DNA Sequencer II (Pharmacia Biotech) was used as a base sequence analyzer.
• a sequencing gel an acrylic amide carrier available as a ready-mix gel (manufactured by Pharmaman Biotech) or a hide-mouth link long ranger (manufactured by FMC) was used.
• A.L.F grade reagents (Pharmacia Biotech) were used as various gel preparation reagents (N, N, K, N'-tetramethylethylenediamine, urea, ammonium persulfate). Nucleotide sequence analysis was performed using an autoread sequencing kit (Pharmacia Biotech). Gel preparation conditions, reaction conditions, and electrophoresis conditions were set with reference to the details of each instruction manual.
• the template DNA, pNCE4Sa1 was alkali-denatured with 10 g of 2 M sodium hydroxide, and then annealed with the universal primer attached to the autoleading-genesis kit to perform an extension reaction. Decoding the reaction product with a sequencer revealed a nucleotide sequence of 546 bp. Based on these results, a FITC-labeled sequencing primer MNEG01 was prepared, reacted with pNCE4Sa1, and further decoded. Based on the obtained results, the following primers were further prepared, and as a result of decoding, the full length of NCE4 was decoded. The prepared FITC-labeled sequencing primer was as shown below.
• FITC-labeled sequencing primers DNA of MNEG-05 to MNEG-08 were synthesized.
• the prepared FITC-labeled sequencing primer was as shown below.
• NEG-05 5'-GACCTGACGGAAGCTGAAGCTCG-3 '(23mer)
• mRNA was prepared from Humicola insolens MN200-1 and cDNA was synthesized by reverse transcription ⁇ , and this was compared with the genomic base sequence to determine the same region.
• Humicola insolens MN200-1 was cultured in a cellulase induction medium, preferably the above (N) medium, for 2 days, and the cells were collected by centrifugation (3500 rpm, 10 minutes). 2 g of the cells were washed with sterile water, and frozen with liquid nitrogen and pulverized with a blender (Nippon Seiki homogenizer AM-3). This was mixed with 10 ml of a denaturing solution containing 4 M guanidine thiosuccinate (4 M guanidine thiocyanate, 25 mM trisodium citrate, 0.5% N-sodium lauryl sarcosine, 0.1 M mercaptoethanol ).
• mRNA Pure Kit manufactured by Falman Abbiotech.
• RNA RNA was dissolved in an lm1 lysis buffer, and subjected to a heat denaturation treatment at 65 ° C for 10 minutes. After quenching in ice, 0.2 ml of sample buffer was added. The entire amount of the RNA solution was charged into an oligo (dT) cellulose column, washed three times with Neusalt buffer and three times with low salt buffer, and eluted with an elimination buffer heated to 65 ° C. . This column operation was repeated twice to obtain an mRNA fraction. The yield was 19.2 g and the yield was 2%.
• cDNA synthesis was carried out using a Timesaver cDNA synthesis kit (Falman Biotech).
• Oligonucleotide primers having the following sequences were prepared as N-terminal and C-terminal primers.
• NCE4-CN 5 '-ATGCGTTCCTCCCCTCTCCTCCGCTCCGCC-3' (30mer)
• NCE4-CC 5 '-TACAGGCACTGATGGTACCAGTCATTAATC-3' (30mer)
• the PCR reaction was performed under the following conditions. First, 1 M of each primer was added to Humicola insolens cDN Al / zg, and heat denaturation was performed at 94 ° (:, 10 minutes in the presence of dNTP. Thereafter, Taq polymerase (recombinant Taq, Takara Shuzo Ne ⁇ 0 was added and amplified by repeating the reaction conditions at 94 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 3 minutes 30. ⁇ The amplified fragment was analyzed by agarose gel electrophoresis and found to be 0.9 kbp. This was concentrated by ethanol precipitation, and cloned with pT7 blue vector kit (Novagen), and this plasmid was designated as pCNCE4.
• the sequencing reaction was performed using the automatic lead sequencing kit as described above. Plasmid p CNCE 4 was alkali-denatured with 2M sodium hydroxide and precipitated with ethanol. This single-stranded plasmid was used as a template and reacted with T7 polymerase. A reaction was performed using the synthetic primers MNEG01, MNEG02 NEG03. MNEG04. MNEG05, MNEG06> MNEG07, and MNEG08, and the universal primer and reverse primer attached to the kit, and the sequence was decoded. As a result, there was one intronic force of 56 bp, '.
• the non-translation initiation sequence, its termination sequence, and the regulatory sequence within the intron are as follows (the numbers are the sequence position numbers of SEQ ID NO: 3).
• LB agar medium containing 100 g / ml ampicillin, lmMI PTG, 0.004% X-ga 1 1% polypeptone, 0.5% yeast extract, l% NaCl, A white colony capable of growing on 1.5% agar was selected and further added to an LB medium containing 100 1111 ampicillin (1% polypeptone, 0.5% yeast extract, l% The cells were cultured at 37 ° C in NaCl). Plasmid DNA was recovered from the resulting culture using the alkaline SDS method.
• This plasmid DNA was cut with BamHI and subjected to 0.8% agarose gel electrophoresis to select a plasmid DNA in which the BaniHI site of PUC118 DNA was disrupted.
• This plasmid DNA was designated as pUC118B.
• pUC118BK DNA was cut with SphI, and a plasmid DNA in which the SphI site of PUC118BN was disrupted was obtained in the same manner as described above.
• This plasmid DNA was designated as pUC118BSN.
• a full-length 3.4 Kbp pstI-XbaI fragment with the sequence was ligated into the PstI-XbaI site of pUC118BSN above.
• the obtained rasmid DNA was designated as PUC118BSN-PX.
• a BamHI site was introduced by site-directed mutation as follows, downstream of the N-terminus of the NCE2 gene and immediately downstream of the stop codon.
• E. coli J Ml09 strain After transforming E. coli J Ml09 strain with plasmid pUC118BSN-PX and further infecting helper phage Ml3KO7, ampicillin and kanamycin were added at 150 g / m1 and 70 ygZm1, respectively.
• the cells were cultured for 16 to 20 hours at 37 in 1 ml of 2xYT liquid medium (1.6% pactotryptone, 0.8% yeast extract, 0.5% NaCi) containing 30 ml 1 at a concentration.
• Ml3 single-stranded DNA (ssDNA) was recovered from the culture supernatant.
• the site-directed mutagenized DNA mixture is introduced into E. coli TGI, and the resulting transformant is transformed into an LB medium containing 100 / m1 ampicillin (1% polypeptone, The cells were cultured in 0.5% yeast extract and 1% NaCi), and plasmid DNA was recovered.
• the plasmid DNA was digested with BamHI, subjected to 0.8% agarose gel electrophoresis, and the plasmid pUC118BSN—PX in which two BamHI sites had been introduced was selected. This plasmid DNA was designated as pM21.
• Genomic DNA of Humicola 'Insolens MN 200-1 was obtained by the method of Example A3.
• the PCR reaction was performed according to the following method according to LA PCR Kit Ver.2 (Takara Shuzo). First, to 1 ⁇ g of Humicola insolens genomic DNA obtained by the above-mentioned method, add 1 400, 400 / ⁇ dNTP, and 2.5 U of LA Taq polymerase to each primer, and add 94UC for 1 minute, 55 ° C for 2 minutes, Amplification was performed by repeating the reaction conditions for 3 minutes for 30 times. 0.8K agarose gel electrophoresis results: 1.6K Amplification of bp DNA was confirmed. The 1.6 Kbp DNA fragment was recovered using a Sephagras band prep kit (Pharmacia Biotech) and ligated to a pT7 blue II-vector kit (Novagen). This plasmid DNA was designated as ⁇ 21.
• Plasmid pK21 DNA was digested with BamHI, and a 1.6 Kbp DNA fragment was recovered.
• plasmid pM21 DNA was digested with BamHI and further treated at 70 ° C for 10 minutes to inactivate the restriction. This was dephosphorylated with calf-derived alkaline phosphatase (Takara Shuzo) and further separated by 0.8% agarose gel electrophoresis to recover a 5.2 Kbp DNA fragment.
• a 1.6 Kb ⁇ DNA fragment derived from PK21 and a 5.2 Kbp DNA fragment derived from ⁇ ⁇ 2 ⁇ were ligated to obtain plasmid pKM04.
• a highly concentrated purified product of pMKD01 was prepared. ⁇ 1 ⁇ : 001 to 5;. Coli J Ml 09 Then, the cells were cultured at 37 ° C. in a 10 Om 1 LB medium containing 100 gZm1 of ampicillin. The resulting culture solution was purified using a Flexiprep kit (Pharmacia Biotech) to obtain 1 ng / 1 pMKD01 plasmid DNA.
• Humicola insolens MN200-1 was cultured in (S) medium at 37 ° C, and after 24 hours, cells were collected by centrifugation at 3000 rpm for 10 minutes.
• the composition of the (S) medium is obtained by adding glucose (3.0%) to the above (N) medium and excluding Abyssel.
• the obtained cells were washed with 0.5 M sucrose and filtered through a 0.45 m filter.
• the protoplast-forming enzyme solution (5 mg / m 1
• Novozyme 234 (NL Ioz
• the protoplasts prepared as described above were suspended in lm1 of SUTC buffer, and 10 ⁇ g of DNA (TE) solution (10 1) was added to 1001 of this, and the mixture was allowed to stand on ice for 5 minutes. .
• a 400 ⁇ 1 PEG solution (60% PEG4000, 10 mM calcium chloride, 1 OmM Tris-HCl (pH 7.5)) was added, and the mixture was allowed to stand on ice for 20 minutes.
• 10 ml of SUTC buffer was added. Centrifuged at 2500 rpm for 10 minutes. After suspending the collected protoplasts in lm1 SUTC buffer, the suspension was centrifuged at 4,000 rpm for 5 minutes and finally suspended in 1001 SUTC buffer.
• Protoplasts treated as above were added to 200 gZm1 of hygromycin.
• plasmid pMKD Ol was introduced into Humicola insolens MN200-1 to select 50 strains showing hygromycin resistance. These were cultured in (N) medium at 37 ° C for 5 days. When the obtained culture supernatant was analyzed by SDS-PAGE, the protein band presumed to be NCE3 was increased by 3 to 4 times from the parent strain in 5 clones among the pMKD O1-transformed strains.
• the N-terminal amino acid sequence of this protein was determined in order to confirm that the expressed protein band was derived from the NCE3 gene.
• the culture supernatants obtained from the parent strain and the NCE3 high expression strain were subjected to column chromatography using an FPLC system according to the method of Example A2, and the main peaks were compared.
• the peak that increased particularly in the NCE3 high expression strain was collected and freeze-dried. This was dissolved in a small amount of water, and electrophoresed using 8% Gel SDS-PAGE mini (manufactured by Tefco).
• the protein was electrically transferred to a PVDF membrane according to the method of Example A2 described above, stained with Koji Brilliant Blue R-250, decolorized, and washed with water. From this, the portion where the protein with a molecular weight of 66 KD was blocked was cut out, and the resulting protein was purified by the method of Podeil, DN et al. (Podell, DN et al., Biochem. Biophys. Res. Commun., 81: 176, 1978), the modified N-terminal residue was removed.
• the target protein is cut out, and incubated at 37 ° C for 30 minutes in a small amount of 0.5% polyvinylpyrrolidone (molecular weight: 40,000, manufactured by Sigma) / 10 OmM ⁇ acid solution. Well washed. next, The modified N-terminal residue was removed with Pfu pyroglutamate aminopeptidase (Takara Shuzo), washed with water, and air-dried. This was supplied to a protein sequencer Model 492, and the N-terminal amino acid sequence was determined at 15 residues. The resulting sequence was as shown below.
• N-terminal amino acid sequence Asn-Cys-Gly-Ser-Leu-Thr-Thr-Glu-Arg-His-Pro-Ser-Leu-Ser-Trp (15 residues)
• Plasmid pMKDOl was digested with BamHI, and the restriction enzyme was inactivated by heat treatment at 70 ° C, followed by dephosphorylation, and an 8.2 Kbp DNA fragment was recovered.
• NCE 4 i3 ⁇ 4F was amplified by the PCR method.
• This PCR product containing NCE4 was designed so that each primer contained a BaraHI site in advance so that it could be ligated in frame with the 8.2 Kbp BamHI fragment of the plasmid pMKDOl.
• Synthetic oligonucleotides having the following sequences were prepared as primers.
• NCE4-N 5- 'CCGGTGTTGGCCGGATCCGCTGATGGCAAG-3' (30mer)
• NCE4-C 5'-TAAGGCCCTCAAGGATCCCTGCGTCTACAG-3 '(30mer)
• the PCR reaction was performed as follows. To 1 g of Humicola insolens genomic DNA, add 1 M each, 400 M dNTPs, and 2.5 U of Pfu DNA polymerase (manufactured by Stratagene) to each 1 g of the primers, and add 94 ° C for 1 minute, 55 ° C for 2 minutes, and 72 minutes. By repeating the reaction conditions at 3 ° C. for 25 minutes 25 times, a 0.8 Kbp DNA fragment was amplified. The 8 Kbp p DNA fragment was recovered and ligated to the 8.2 Kb p BamE I fragment of pMKD01 described above. This plasmid DNA was designated as pEGDO1.
• the N-terminal amino acid sequence of this protein was determined in order to confirm that the expressed protein band was derived from the NCE4 gene.
• the culture supernatant obtained from the parent strain and the NCE4 high-expressing strain was subjected to column chromatography using an FPLC system, and the main peaks were compared. The conditions were the same as in Example B2 above.
• the peak which particularly increased in the NCE4 high expression strain was collected and freeze-dried. This was dissolved in a small amount of water. After removing the modified N-terminal residue according to the method of Example B2, the N-terminal amino acid sequence was determined by the aforementioned protein sequencer.
• N-terminal amino acid sequences were obtained at a ratio of about 7: 3.
• the resulting sequence was as shown below.
• the amino acid sequence at the N-terminal was determined using the above-mentioned protein sequencer without removing the modified N-terminal. As a result, only the amino acid sequence 1 shown below was obtained.
• N-terminal amino acid sequence 1 Val-Val-Glu-Glu-Arg-Gln-Asn-Cys-Gly-Ser-Ala-Asp-Gly-Lys-Ser-Thr-Arg-Tyr-Trp-Asp (20 residues )
• N-terminal amino acid sequence 2 Asn- (Cys) -Gly-Ser-Ala-Asp-Gly-Lys-Ser-Thr-Arg-Tyr-Trp-Asp- (Cys)-(Cys) -Lys-Pro- Ser- (Cys) (20 residues)
• N-terminal amino acid sequences matched the amino acid sequences of the cellulase NCE2 and NCE4 fusion proteins deduced from the nucleotide sequence of plasmid pEGD01. The fact that two types of N-terminal amino acid sequences were obtained revealed that the fusion protein was processed at multiple positions when the signal sequence was cleaved.
• Humicola Insolens MN2 00-1 (parent stock) 0.28 g
• Production volume is the production volume per 1 L of culture solution. ⁇ ⁇
• Example B5 Plasmid p I £ 002
• Plasmid pEGD01 was digested with HindIII and BamHI to recover a 7.2 Kbp pNA fragment.
• NCE1 ⁇ derived from Humicola insolens obtained by the method described in JP-A-8-5663
• the promoter and signal sequence of NCE1 ⁇ were copied by PCR.
• the portion of the DNA to be loaded was widened.
• the PCR product containing the NCE1 promoter and the signal sequence was previously provided with HindIII and BamHI sites in each primer so that it could be ligated to the 7.2 kb HindIII to BamHI fragment of the plasmid pEGD01. Designed to include A synthetic oligonucleotide having the following sequence was used as a primer.
• PNCE1-N 5 '-GTCATGAAGCTTCATTAAGGTACGTATGCAAC-3' (32mer)
• PNCE1-C 5'-GGTGATGGATCCGGCCTGCTGGGCAGCGACGC-3 '(32mer)
• the PCR reaction was performed in the same manner as in Example 3.
• This PCR product was digested with HindIII and BamHI to recover a 1.5 Kbp DNA fragment. This was ligated to the 7.2 Kbp HindIII to BamHI fragment of pEGD01 described above. This The plasmid DNA was designated as pID01.
• Plasmid pID01 was digested with BamHI, the restriction fragment was inactivated by heat treatment at 70 ° C, and further dephosphorylated, after which a 8.6 Kbp DNA fragment was recovered.
• the plasmid pEGDO1 was digested with BamHI, and a 0.8 Kbp DNA fragment containing NCE4 fragment fc ⁇ was recovered. The two fragments were ligated to obtain plasmid pIED02.
• Example B6 Expression of plasmid pI EDO2
• the N-terminal amino acid sequence of this protein was determined in order to confirm that the expressed protein band was derived from the NCE4 gene.
• the culture supernatant obtained from the parent strain and the NCE4 high-expressing strain was subjected to column chromatography using an FPLC system, and the NCE4 peak was collected and lyophilized. did. Dissolve this in a small amount of water did.
• 15 residues of the N-terminal amino acid sequence were determined by the aforementioned protein sequencer. The resulting sequence was as shown below.
• N-terminal amino acid sequence Gln-Ala-Gly-Ser-Ala-Asp-Gly-Lys-Ser-Thr-Arg-Tyr-Trp
• This N-terminal amino acid sequence coincided with the amino acid sequence of the cellulase NCE1 and NCE4 fusion protein deduced from the nucleotide sequence of plasmid pIDE02.
• Production volume is the production volume per 1 L of culture solution ⁇
• Organism name Humicola insolens
• Sequence type nucleic acid
• Organism name Humicola insolens
• ATC GTC GAC CAG GGC CGC AGC GGC AAG CAG CCC ACC GGC CAG AAG GAA 1737 lie Val Asp Gin Gly Arg Ser Gly Lys Gin Pro Thr Gly Gin Lys Glu
• Sequence type nucleic acid
• Organism name Humicola insoiens
• Fragment type N-terminal fragment origin
• Fragment type N-terminal fragment
• Organism name Humicola insolens
• Fragment type N-terminal fragment
• Fragment type N-terminal fragment
• Organism name Humicola insolens

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